Characterization of partially purified 8 kDa antigenic protein of Clonorchis sinensis

Abstract

The 8 kDa antigenic protein of Clonorchis sinensis was partially purified by ammonium sulfate precipitation and subsequently by a column chromatographic steps. The purified protein was separated into 7 and 8 kDa protein bands through SDS-tricine gel electrophoresis, while the protein was found to migrate to a 8 kDa band in 7.5-15% SDS-PAGE. The molecular weight of the antigen was estimated to be 110 kDa by Superose 6 HR 10/30 gel filtration. The purified antigen strongly reacted with the human sera of clonorchiasis. The hyperimmune sera of BALB/c mice immunized against the 8 kDa protein were reacted with both the crude extract and the excretory-secretory product of adult worms, but not with the metacercarial extract. Immunohistochemical staining demonstrated that the protein was distributed to the tegument and subtegumental cells and also to the seminal receptacle. The present findings suggest that the 8 kDa protein is a partition of the multicomplex protein originating from various organs of adult C. sinensis, and that it is composed of several 7 and 8 kDa proteins.

Fig. 1

Purification of 8 kDa protein from Clonorchis sinensis crude extracts. A, Purified 8 kDa protein was analyzed on 7.5-15% gradient SDS-PAGE. Lane C, crude extracts; lane 1, supernatant of ammonium sulfate saturation; lane 2, partially purified 8 kDa fraction through Sephacryl S-100 HR column; lane 3, purified 8 kDa protein. B, Analysis of the purified protein by SDS-tricine system. Lane C, crude extracts; lane 1, partially purified 8 kDa; P, purified 8 kDa. The 8 kDa protein was separated to 7 and 8 kDa proteins (arrows). C, Superose 6 HR 10/30 gel filtration of partial purified 8 kDa protein. The 8 kDa protein was eluted at fraction of volume 15.5 ml (indicated by bar). D, Estimation of molecular weight of the 8 kDa protein. The protein was estimated to be 110 kDa through the Superose 6 HR column. Standard marker proteins were capitalized. A, β-amylase (200 kDa); B, alcohol dehydrogenase (150 kDa); C, bovine serum albumin (66 kDa); D, cytochrome C (12.4 kDa).

Fig. 2

Immunoblotting of crude extracts and 8 kDa protein with clonorchiasis sera. A, Crude extracts of C. sinensis. B, Purified 8 kDa. Electrophoresis was performed on 7.5-15% gradient gel.

Fig. 3

A, Production of anti-8 kDa polyclonal antibody. BALB/c mice were used as described in materials and methods. B, Immunoblotting of crude extracts of metacercariae, adult and adult ESP with anti-8 kDa polyclonal antibody. The antibody was able to recognize the 8 kDa protein of adult and ESP, but not that of metacercariae.

Fig. 4

Reactivity of anti-8 kDa polyclonal antibody with proteins of human-infecting other parasites. The antibody demonstrated cross-reaction with crude extracts of Fasciola hepatica.

Fig. 5

Immunolocalization of 8 kDa protein with immune serum. A and C, The protein was distributed at the tegument and subtegumental cells (arrows). Eggs were also slightly reacted with the anti-8 kDa antibody. Right panels B & D showed the same slide with hematoxylin-eosin staining. E, Seminal receptacle of the worm was also strongly reacted with the antibody. F, Hematoxylin-eosin staining of the slide E.